Cytotoxicity of 212Pb-labeled anti-PTK7 antibody in 2D adherent and 3D multicellular bladder cancer models

Abstract

Background: Bladder cancer remains a significant global health challenge, with approximately 75% of cases presenting as non-muscle-invasive bladder cancer. Despite standard treatment with transurethral resection and intravesical Bacillus Calmette-Guérin immunotherapy, up to 40% of patients develop resistance or progress to muscle-invasive disease. Targeted alpha-emitting radionuclide therapy offers promising therapeutic potential through the selective delivery of high linear energy transfer radiation to tumor cells while minimizing damage to healthy tissues. PTK7 is overexpressed in various malignancies, including bladder cancer, and is therefore a viable therapeutic target. This study evaluated the preclinical efficacy of [²¹²Pb]Pb-TCMC-chOI-1, a ²¹²Pb-labeled antibody targeting PTK7, for targeted alpha-emitting radionuclide therapy in bladder cancer using 2D adherent cultures (clonogenic assay) and 3D multicellular spheroid models (spheroid growth inhibition).

Results: PTK7 expression analysis revealed varying antigen densities across five bladder cancer cell lines, ranging from approximately 10,000 to 70,000 sites per cell. The chimeric anti-PTK7 antibody demonstrated apparent equilibrium dissociation constants of 10-44 nM with moderate binding affinity suitable for therapeutic applications. [²¹²Pb]Pb-TCMC-chOI-1 treatment resulted in activity- and time-dependent cytotoxicity, with enhanced sensitivity observed in cell lines with higher PTK7 levels. In clonogenic assays, the activity concentration required for 50% growth reduction was 48-74 kBq/mL, corresponding to 22-51 bound and 9-16 internalized ²¹²Pb atoms per cell. In 3D models, similar therapeutic effects were observed despite significantly lower activities (values of approximately 1 and 30 kBq/mL for KU-19-19 and 647-V cells, respectively), suggesting a more pronounced cross-fire effect. Flow cytometry demonstrated treatment-induced DNA damage, cell cycle perturbations and cell death, with response patterns correlating with overall treatment sensitivity. RT-112 and KU-19-19 cells showed superior responses compared to 647-V and T-24 cells, consistent with their higher PTK7 expression.

Conclusions: These findings support PTK7 as a therapeutic target for bladder cancer and demonstrate the potential of [²¹²Pb]Pb-TCMC-chOI-1 for targeted alpha-emitting radionuclide therapy. The results provide a rationale for further preclinical optimization of this therapeutic approach. Trial registration number (TRN): Not applicable.

Keywords: 212Pb; Bladder cancer; Internalization; Monoclonal antibody; PTK7 (Protein tyrosine kinase 7); Targeted alpha therapy (TAT).

Fig. 1

Binding and internalization of [²¹²Pb]Pb-TCMC-chOI-1 and [²¹²Pb]Pb-TCMC-hlgG in bladder cancer cells over time. Cells were incubated with 10 kBq/ml [²¹²Pb]Pb-TCMC-chOI-1 or [²¹²Pb]Pb-TCMC-hIgG1 (both at 50 MBq/mg) for 1, 4, and 24 h at 37 °C. After incubation, the cells were washed and treated with a stripping buffer containing trypsin for 1 h at 37 °C to remove the surface-bound ²¹²Pb-TCMC-mAbs. The soluble fraction (cell-surface-bound) and cell pellet (internalized) were collected after additional washes were performed. Cell surface-bound and internalized activities were measured using the gamma counter. A) Surface-bound and B) internalized ²¹²Pb atoms per cell. The results are presented as the average of three to four biological experiments with error bars. P-values were calculated using an unpaired t-test with correction for multiple comparisons using the Holm-Sidak method (*p < 0.05, **p < 0.005, ***p < 0.0005)

Fig. 2

Clonogenic fractions of bladder cancer cell lines treated with [²¹²Pb]Pb-TCMC-chOI-1. A) Clonogenic fractions of RT-112, KU-19–19, 647-V, and T-24 cells treated with 80 kBq/mL [²¹²Pb]Pb-TCMC-chOI-1 of varying specific activities (5 MBq/mg or 50 MBq/mg) or isotype control ([²¹²Pb]Pb-TCMC-hIgG) across incubation times of 1, 4, and 24 h; The black line above the bars (—) compares 50 MBq/mg with both 5 MBq/mg and the isotype control group. B) Clonogenic fractions as a function of activity concentration (0–80 kBq/mL), total, or internalized ²¹²Pb atoms/cell after a fixed incubation time of 4 h fitted using the single-hit model (Clonogenic fraction = exp(− A/A₀), where A is the activity (kBq/mL), and A₀ is the activity to reduce survival by 67%. C) The IC₅₀ values of activity concentration, and surface bound and internalized ²¹²Pb atoms per cell were plotted against the number of antigen sites per cell. Statistical significance was determined using an unpaired t-test with Holm-Sidak correction (*p < 0.05). Results are presented as mean ± SD from three independent experiments

Fig. 3

Growth inhibition of bladder cancer spheroids treated with [²¹²Pb]Pb-TCMC-mAbs. A Growth curves showing spheroid size over time for KU-19–19 (left) and 647-V (right) spheroids treated with radiolabeled [²¹²Pb]Pb-TCMC-chOI-1 or [²¹²Pb]Pb-TCMC-hIgG (isotype control = IC, blue line) at different activity concentrations for incubation periods of 1, 4, and 24 h. The orange line indicates pre-treatment with chOI-1 for 30 min. The control consisted of spheroids maintained in the cell medium. B Representative brightfield and fluorescent images of KU-19-19 and 647-V spheroids treated with control or 50 kBq/mL radiolabeled antibody for 24 h. Spheroids were stained with fluorescein diacetate (FDA; green channel for live cells) and propidium iodide (PI; red channel for dead cells). The merged images show the live/dead cell distributions within the spheroids over time. Scale bars = 200 µm

Fig. 4

Cell viability, apoptosis, DNA damage, and mitotic analysis in KU-19-19 and 647-V cells following [²¹²Pb]Pb-TCMC-chOI-1 treatment. A Quantification of apoptotic, necrotic, dead, mitotic, and γH2AX-positive (DNA damage) fractions in KU-19-19 and 647-V BC cells after exposure to 100 kBq/mL [²¹²Pb]Pb-TCMC-chOI-1 for 24 h. Control refers to cells left in the cell medium. For apoptosis and cell death analysis, cells were harvested on days 1, 3, and 6 post-treatment, stained with Annexin V-FITC and propidium iodide, and analyzed using flow cytometry (CytoFLEX S, CytExpert software). To assess mitotic cells and DNA double-strand breaks, collected cells were stained with FVD-eFluor450, anti-pS10H3/AlexaFluor 647 (mitosis), anti-γH2AX/FITC (DNA damage), and propidium iodide for the flow analysis. All fractions are presented as mean ± SD from three independent experiments performed in triplicates. DNA damage is presented as fold change compared to that in control cells. Statistical significance was determined using an unpaired t-test with Holm-Sidak correction for multiple comparisons (*p < 0.05, **p < 0.005, ***p < 0.0005). B Representative flow cytometry plots showing untreated 647-V cells (left) and cells treated with 100 kBq/mL of [212Pb]Pb-TCMC-chOI-1 (right) on day 6 after treatment

Fig. 5

Cell cycle progression of KU-19-19 and 647-V after treatment with [²¹²Pb]Pb-TCMC-chOI-1 A Cell cycle distribution of viable KU-19-19 and 647-V cells following treatment with 100 kBq/mL of [²¹²Pb]Pb-TCMC-chOI-1 for 24 h. Cells were harvested 24 h post-treatment and stained with FVD-eFluor450 and propidium iodide. Control refers to cells left in the cell medium. B) Representative DNA histograms showing untreated 647-V cells (upper) and cells treated with 100 kBq/mL of [²¹²Pb]Pb-TCMC-chOI-1 (lower) 24 h post-treatment. Samples were analyzed using a CytoFlex S Flow Cytometer and CytExpert software. Cell cycle distributions are expressed as mean ± SD from three independent experiments performed in triplicate. P-values were calculated using an unpaired t-test with correction for multiple comparisons using the Holm-Sidak method (**p < 0.005, ***p < 0.0005)